Anti-inflammatory, immunomodulatory and anti-oxidant effects of Ocimum basilicum L. and its main constituents: A review

Ocimum basilicum L. (O. basilicum) is an ornamental and therapeutic plant with various pharmacological effects and medical applications. In this article, detailed information on the anti-oxidant, immunomodulatory, and anti-inﬂammatory properties of O. basilicum and its main constituents was provided. The literature survey of the different databases until the end of November 2021 was explored on the immunomodulatory, anti-inﬂammatory and anti-oxidant effects of the herb and its constituents. The plant and its constituents showed diverse pharmacological effects including immunomodulatory, anti-inflammatory and anti-oxidant properties by improving of the inflammatory mediators including interleukin (IL)-10, IL-4, tumor necrosis factor-alpha (TNF-α), interferon gamma (IFN-γ), nitric oxide (NO), serum levels of IFN-γ, IL10 and IL-4, Ig. G, Ig. M and phospholipase A2 (PLA2), immunoglobulin E (Ig. E), total protein (TP), oxidant and anti-oxidant markers. O. basilicum and its main constituents therefore, could be effective on the treatment of diseases associated with inflammation, immune dysregulation and oxidative stress. The present review article provides readers with organized information about the anti-oxidant, immunomodulatory, and anti-inﬂammatory properties of O. basilicum.


Introduction
The use of the natural materials of herbs by humans for diverse aims such as treatment of different diseases could be come back to the cave dwelling era. Traditional plants exert important role in discovery of new drugs. Medicinal plants, namely herbal medicine, are the most of bio resource of drugs for modern and traditional medicines, food supplements, nutraceuticals, folk medicines, and pharmaceutical intermediates. It was shown that out of 3,000 known plant species in the world, only 15% of them are used for medicine potential (1,2). Recognizing the importance of plants in discovering new and safer therapeutic agents, screening of medicinal plants in terms of medicinal activities and phytochemical compounds is one of the active research fields worldwide (3). and several plants have been broadly used for their pharmacology and medicinal aspects (4). A plant contains a plenty of various molecules that may act synergistically on targeted elements of the complex cellular pathway so plants are essential source for medicinal compositions (5). Aromatic plants have been used for home treatment, subsistence, and traditional therapies and are used for local livelihoods and income generation (6). These plants have many applications at different fields such as food flavoring industries, cosmetic industries, alcoholic beverages, soft drinks, and pharmaceutical industries. Herbs as aromatic plants contain phytonutrients which can be used as a flavoring foods or beverage and most importantly used in the treatment of many diseases (7). The Ocimum basilicum L. (O. basilicum) is a plentiful source of polyphenolics and represents high diversity (8). The genus Ocimum belongs to the family Lamiaceae and various species of Ocimum are known that are used to treat various types of illnesses from old time, chiefly the species O. basilicum (Taxonomical Hierarchy of O. basilicum are shown in Figure 1). The O. basilicum is one of the most used plant that generally known as Sweet Basil which is found in tropical and subtropical regions of Africa, Asia, and south America (9). The O. basilicum leaves may taste like anise, via a strong, pungent, and sweet smell. The flowers of this plant are small, purple/white in color and it is generally removed to increase leaf yield (10, 11) ( Figure  2). The fresh O. basilicum is usually used in cooking recipes and the plant species of this genus have many potentials in medical application. Nowadays, the interest in bioactive compounds of the Basil (O. basilicum) has increased due to its long-standing usage in folk medicine, especially antiinflammatory, immunomodulatory, anti-oxidative, and anti-microbial properties. The O. basilicum is a herb that has been used in traditional Indian and Asian medicine for thousands years as a natural antibiotic, anti-inflammatory, analgesic, and diuretic (12,13). Due to its chemical composition, it is used in cooking, perfumery, toothpaste, food industry, production of cosmetics and medicine (14). Several studies were focused their attention on this plant which is native in all part of the Mediterranean and widely commercially planted in Iran. The biological activity of O. basilicum, characterized it in various pharmacology, traditional and medicinal usage (15). This plant contains a wide range of natural products including polyphenols such as anthocyanins and flavonoids (16). This review article aimed to optimize and validate the phytochemical study and the anti-inflammatory, immunomodulatory, antiinflammatory, and anti-oxidant effects of O. basilicum and its main constituents.

Methods
The online publications were checked using variant search engine such as Google scholar, Scopus, PubMed, Web of Science, and Science direct. The main keywords used for searching were: Ocimum basilicum, sweet basil, linalool, eugenol, rosmarinic acid, polyphenol, flavonoid, essential oil, anti-oxidant, anti-inflammatory, and immunomodulatory in addition to related keywords alone and combined with each other. The published papers from 1998 to end of November 2021 in English language were included in the review.
basilicum contain high concentration of minerals, vitamins , and oils (27). The essential oil of the flowers of this plant contain small amount of estragol, eucalyptol, ocimene, linalool, acetate, eugenol, 1-epibicyclosesquiphellandrene, menthone, methanol, cyclohexanol, cyclohexanone, nerol, and myrcenol (28). A research showed that the seeds of O. basilicum contain a gum which have major fractions of glucomannan with (1⟶4)-linked xylan and a minor fraction of glucan (29). Some example of the O. basilicum essential oil chemical ingredients in different parts are shown in Table 1 and the chemical structures of some compound are displayed in Table 2.

Anti-inflammatory effect
Inflammatory cells such as mast cells, T lymphocytes, and eosinophils have a main role in the pathogenesis of immune-mediated diseases (30,31). Most of the human population is getting affected by inflammation related Table 1. Some examples of chemical composition of Ocimum basilicum essential oil in various parts of plant disorders. Therefore, anti-inflammatory agents could be of therapeutic value in the treatment of inflammatory diseases. The anti-inflammatory property of O. basilicum and its derivatives was shown in several studies.

Anti-inflammatory effect of O. basilicum extracts and essential oil
The anti-inflammatory properties of the O. basilicum essential oil complexed with β -cyclodextrin (OBEO/β-CD) in mice models of paw edema induced by carrageenan showed that conjugation of β-cyclodextrin with O. basilicum inhibited the number of total lymphocytes, leukocytes, granulocytes and monocytes in the abdominal cavity of carrageenan-induced animals, indicating the effectiveness of this complex in regulating leukocyte recruitment during an acute inflammatory response. The findings suggest that this plant can be used for the production of an anti-inflammatory drug (32). Various chemical elicitors such as jasmonic acid (JA), arachidonic acid (AA), and baminobutyricacid (BABA) can cause changes in the phenolic levels a then the anti-inflammatory potential of purple O. basilicum leaves. The results showed all tested elicitors increase the amounts of phenolic ingredients including phenolic acids and flavonoids and cause the highest anti-inflammatory activities in comparison with control group. Anthocyanin contents in O. basilicum is about 0.1 mg/g. The level of anthocyanins in its leaves has been significantly enhanced after stimulation with abiotic elicitors such as jasmonic acid, arachidonic acid and b-aminobutyric acid. Anthocyanins have ability to inhibition of lipoxygenase (LOX) activity and act as anti-inflammatory agents. The stimulation of basil by all of the elicitors enhanced its ability to inhibit the LOX activity, expressed as an IC 50 value. LOX is responsible for the metabolism of the fatty acids and their metabolites eliciting inflammatory responses in the body (33). The analgesic effects of the O. basilicum essential oil in the inflammatory pain models in mice indicated that the analgesic effects of essential oil of the O. basilicum is similar to the linalool and eugenol which were mediated by mu-and delta -opioid pathways and further express that O. basilicum essential oil has a potential use as an analgesic agent for the alleviation of inflammatory pain (34). The anti-inflammatory effect of the O. basilicum extract against inflammation induced by adipocyte, possibly through suppression of Tnfrsf9 (TNF receptor superfamily member) has been shown (35). High O. basilicum extract concentration also decreased total WBC count and significantly increased anti-inflammatory and anti-oxidant parameters in the asthmatic rats similar to the dexamethasone (36). In a study, the chemical composition and systemic anti-inflammatory activity of the O. basilicum essential oil was investigated and verified via acute and chronic in vivo experiments as peritonitis, paw edema, vascular permeability, and granulomatous inflammation model by the participation of histamine and arachidonic acid pathways. The results demonstrate that the essential oil has more efficacious in the acute and chronic antiinflammatory action. This research approves the therapeutic potential of O. basilicum and reinforces the validity of its use in the general medicine (37). In another study, the inhibitory effect of the O. basilicum extract on the key pro-inflammatory mediators and cytokines have been shown, which accounts for its anti-inflammatory effect (38,39).

Anti-inflammatory effect of O. basilicum constituents
It was shown that mucilage of this plant could apply more protection against inflammatory mediators and oxidative stress in colitis suggesting this agent as a good candidate for colitis treatment as complementary therapy. Anti-inflammatory effect on colitis might be attributed to the presence of terpenoids and flavonoids which are known to inhibit the inflammatory signaling through NF-kB suppression. NF-kB through TNF-alpha activation has a pivotal role in the IBD pathology and recurrence and as its suppression could result in colitis remission both in the experimental and clinical settings. Besides, it revealed that treatment with this plant decrease the wet weight of distal colon segments and major damage score in comparison with the control which is well correlated with regression in local inflammation scores (40). In another study, the major ingredients of the residue fraction of O. basilicum were shown as methyl eugenol (11.35%), estragole (17.06%) and linoleic acid (11.40%), while the distillate fraction primarily contained a-cadinol (16.24%), methyl eugenol (16.96%)   Table 3.

Anti-oxidant effect
Living cells of the organisms are involve in oxidative reactions for many goals including metabolism, cell communication, death or renewal, and defense mechanisms (42). Oxidizing agents and free radicals containing one or more unpaired electrons in their outer orbit that makes them reactive species namely electrophiles. The reactions of these free radicals with the organisms causes damage of the cell and tissue. The free radicals act on cellular components by oxidizing proteins, lipids, carbohydrates, and nucleic acids. The free radicals oxidized cellular components such as proteins, lipids, carbohydrates, and nucleic acids (43,44). Anti-oxidants have a significant role in hampering a diversity of lifestyle-related diseases and aging so these are associated with active oxygen and lipid per-oxidation (45,46). Herbal medicines applicate their anti-oxidant effects by multiple mechanisms (47)(48)(49)(50).

Anti-oxidant effect of O. basilicum extracts and essential oil
The anti-oxidant activities of the herbal extracts is mainly due to their capacity to be donors of hydrogen or electrons and to capture the free radicals (51). In cultivars using online acidic potassium permanganate chemiluminescence total phenolics, anti-oxidant content, and individual polyphenolics of the extracts from O. basilicum were examined. The results showed high polyphenolic producing in Red Rubin, Holy Green, and Basil Genovese low amount in Subja. Therefore, anti-oxidant potential was more potent in the purple flowered plant than its white flowered type (12). It was indicated that the extract of the O. basilicum acts as an anti-oxidant (52,53) and effectively subjugates the effects of high oxidizing agents such as hydrogen peroxide (54, 55). These actions are attributed to its composition, which is rich in flavonoids and polyphenols as well as compounds such as rosmarinic acid (RA), all of which have well-known to show anti-oxidant property (43). The anti-oxidant potential of methanolic extract of O. basillicum was studied using DPPH assay (60). Intake of the O. basilicum extract may decrease hepato-renal toxicity induced by acetaminophen (61) that attributed to its antioxidant property and may be due to suppression of liver lipid synthesis. O. basilicum administration modulated both kidney and liver pathological changes confirming the protective role of O. basilicum against hepatic/nephron toxicity and oxidative injury caused by paracetamol overdose (62). The better anti-inflammatory and antioxidant potential of the combination of essential oils of the O. basilicum/O. gratissimum occurs by inhibition of all isoforms of the cyclooxygenase by the inhibition of ≥ 98% for cyclooxygenase 1 and ≥ 67% for cyclooxygenase 2  (63). The essential oils of the O. basilicum indicated antioxidant and free radical-scavenging activities. In a study, the effect of growing season on chemical composition, antimicrobial and anti-oxidant activities of the essential oils from O. basilicum was investigated. The results revealed that in winter oxygenated monoterpenes were found to be richer, while those of summer were higher in sesquiterpene hydrocarbons. Linalool, the major component of the O. basilicum essential oil, exhibited lower anti-oxidant activity than the entire oil. The anti-oxidant activity of essential oils of the O. basilicum also might be attributed to the presence of other phenolic compounds (64). Essential oils of Iraqi growing O. basilicum contained linalool (48.69%), transα-bergamotene (8.23%), 1,8-cineole (14.00%) and eugenol (6.64%). The anti-oxidant investigation displayed strong inhibition of 110.8% against autoxidation by linoleic acid, while the scavenging of the DPPH radical gave a value of IC50 145.35 μg/mL. The findings showed that these essential oils could be used for pharmaceutical works and preservative in the food industry (65).
The use of the O. basilicum leaves powder as broilers chicken feed additive can ameliorate the anti-oxidant activity of broilers dose dependently so that its high concentrations causes increasing the serum CAT enzyme and reducing MDA levels (66). Improvement effect of O. basilicum essential oil on acetic acid-induced colitis in rats was investigated. Higher concentrations of O. basilicum essential oil (200 and 400 mL/kg) significantly reduced severity, area, and index of ulcer. Treatment with the essential oil of this plant also decreased the level of myeloperoxidase in colitis. These findings suggest that O. basilicum exhibits protective effect against acetic acid-induced colitis (67). A study was shown the anti-platelet aggregation effect of aqueous extract of O. basilicum. The plant extract suppressed the elevated vascular contractions induced by HCD and inhibited ADPinduced platelet aggregation. Thrombin-induced platelet activation was decreased by 15%, 23%, 40%, 38.4%, and 42% at the same doses of the extract. In hypercholesterolemia, one of the main causes of the lowered function of endothelial cells can be an enhance of superoxide release. Fatty acid and triglyceride-rich emulsions can stimulate leukocytes to generate ROS which are very toxic against the vascular wall cells (68)W¦X.

Anti-oxidant effect of O. basilicum constituents
Different parts of the O. basilicum like flowers, leaves and roots were found to be rich reserves of the anti-oxidant compounds. Anti-oxidant activity could be varied by the levels of flavonoids and phenols in O. basilicum (69). The polyphenols such as chicoric, rosmarinic, m-coumaric, caffeic, p-coumaric, and ferulic acids content of this plant exhibited anti-oxidant activity (70). The effects of the Rosmarinus acid (RA) in the ovalbumin sensitized rats showed that treatment of the sensitized animals with RA decreased cholesterol synthesis and lipid accumulation in human macrophages (56). Various therapeutic effects have been explained for RA such as anti-inflammatory, analgesic, immunomodulatory actions, and antibacterial activities. In a study, the effect of RA on inhibition of gentamicin-induced nephrotoxicity in rats and showed that RA reduced gentamicin nephrotoxicity via anti-oxidant activity by increase the renal GSH content and other antioxidant enzymes activities (71). Eugenol, carvacrol, thymol, and 4-allylphenol displayed better anti-oxidant activities than the other components of the plant. These components inhibited the oxidation of hexanal approximately 100% for 30 days and a concentration of 5μg/ml (72). In another research, the effect of RA on lung inflammatory cells, tracheal responsiveness (TR), and oxidant markers in the sensitized rats compared to the dexamethasone were evaluated and data indicated the dose-dependent effect of RA on tracheal responsiveness, inflammatory and oxidant-antioxidant parameters. Treated with this plant showed that antioxidant biomarkers (thiol, SOD and CAT) and percentage of lymphocyte were significantly less than those of control (56).
Anti-oxidant effects of the O. basilicum and its constituents are shown in Table 4.

Immunomodulatory effect
Immune system dysfunction can cause serious diseases. Immunomodulatory agents are able to improve and overcome many barriers to treatment methods, such as off-target side effects, inadequate immune stimulation, and bioactivity loss of immune agent. Immunotherapy means treating a disease by suppressing or activating immune system (73). Immune pathways driven by T-helper 2 (Th2) cells which produce IL-5, IL-4, and IL-13 can involve in the pathophysiology of many inflammatory diseases. The imbalance of Th1/Th2 toward increased Th2 activity was shown in the pathogenesis of allergies (9,74). Th2 cells release cytokines such as IL-4 and IL-5 that leads to the production of IgE by B cells and therefore they have an important role in allergy. In fact, IL-4 causes excessive production of immunoglobulin E (IgE), IL-5, activates eosinophils, and IL-13 which lead to mucus hyper-secretion (75)(76)(77). Immune and inflammatory cells such as dendritic cells, leukocytes, and T cells, as well as cytokines, play important roles in immune reactions (78). Flavonoids, triterpenes, and catechins are biologically active components of the plants that play a main role in immunoregulatory activity (79).

Immunomodulatory effect of O. basilicum extracts and essential oil
Hydro-ethanolic extract of O. basilicum showed the antiinflammatory effects on lung pathological changes in OVAinduced asthma. Treatment with O. basilicum extract lead to decrease IgE, IL-4, phospholipase A2 (PLA2) and total protein (TP) levels but increase interferon gamma (IFN-γ) and IFN-γ/IL-4 ratio in comparison with the healthy rats. The plant significantly improved the pathological changes of the sensitized rats (80). Also, in the similar model, O. basilicum extract decreased total WBC count, percentages of monocytes, eosinophils, neutrophils, and levels of oxidant markers compared to the untreated sensitized rats (36). In another study, the anti-inflammatory activity of the O. basilicum extracts using peripheral blood mononuclear cells (PBMC) of the healthy individuals showed that methanolic extract of the O. basilicum inhibits the key proinflammatory cytokines like IL-1β, IL-2, TNF-α, and other mediators, which accounts for its anti-inflammatory effect (38). The methanolic extract from this plant can inhibits platelet aggregation induced by collagen and it has the most potent inhibitory effects on platelet aggregation induced by ADP (81) and also showed inhibitory activity to counter HIV-1 reverse transcriptase (82). The immune effects of O. basilicum have been reported previously (83). Farida et al. showed that administration of O. basilicum leaves powder as a feed additive in chicken can boost the innate and adaptive immune response to Newcastle disease virus (NDV) vaccine (66). The effect of O. basilicum oil on the non-specific immune response of Niletilapia (Oreochromis Nilotic us) and resistance against some aquaculture disease such as Aeromonas infection was also demonstrated (84). The direct immunomodulatory effect of extract of the O. basilicum on human immune cells by Th2 and Th1 cells as well as regulatory T derived cytokines, possibly via the ERK2 signal pathway was also reported. This plant could be used for treating immune dysregulatory disorders. Extract of the O. basilicum suppressed cytokines produced by Th1 (IL-2, TNF-β, and IFN-γ), Th2 (IL-10, IL-5) as well as regulatory T (TGF-β) cells, and expression of ERK2 mRNA in peripheral blood mononuclear cells (PBMC) and also suppressed some of the cytokines. Therefore, the results indicate that O. basilicum has direct immunomodulatory effect on basic functional properties of human immune cells, possibly mediated by the ERK2, MAP-kinase signal pathway (85). The extract of O. basilicum leaves on animal model of asthma increased the IFN-γ/IL-4 ratio (Th1/Th2 balance) but decreased bronchoalveolar lavage fluid (BALF) levels of IgE, phospholipases A2 (sPLA2), and TP (9). The effect of aqueous extract of the O. basilicum at 31.25, 62.5, 125 and 250 mg/ml concentrations on lymphocyte proliferation showed 36 and 80% decrease in proliferation of resident lymphocytes at 125 and 250 mg/ml respectively (86). In a study the cytoprotective effects of RA from O. basilicum against mycotoxin, aflatoxin, and ochratoxin was shown by dose dependently inhibition of DNA and protein synthesis. Apoptosis was prevented by decline of DNA fragmentation and inhibition of caspase-3 (87). Ethanolic extract of O. basilicum and earning flavonoid content at 400 mg/kg/day in mice showed an increase in circulating antibody titer in response to sheep red blood cells (SRBCs) and also increased the percentage neutrophils adhesion to nylon fibers and phagocytic activity (88).

Immunomodulatory effect of O. basilicum constituents
Compounds extracted from O. basilicum showed immunomodulatory action happening in the cellular level. In a study, it has been shown that constituent of O. basilicum reduced IL-4, IgE, PLA2 and TP levels, but enhanced IFN-γ/IL-4 ratio and therefore affected lung pathological changes (80). The flavonoid content of this plant can produce a marked increase in percentage neutrophils adhesion to nylon fibers and phagocytic activity and show immunostimulant effect (89). Immunomodulatory effect of O. basilicum and its constituents are summarized in Table 5.

Discussion
The extensive survey of literature displayed that O. basilicum has an enormous spectrum of pharmacological activities. The current review is meant to describe the importance of O. basilicum in the field of herbal medication. The effect of O. basilicum and its constituents on inflammation, oxidative stress and immune system were reviewed. Crude extracts and essential oil of different parts of this plant have been used for their various effects like anti-inflammatory, immunomodulatory, anti-oxidant, due to its bioactive phytocomponents. According to the results of several studies using various models, this plant and its constituents such as polysaccharides, phenolics and flavonoids indicated anti-oxidant, anti-inflammatory, 5 Table 5. Immunomodulatory effect of Ocimum basilicum and its constituents PBMC: Peripheral blood mononuclear cells, TNF-α: Tumor necrosis factor-α, IL: Interleukin, SRBCs: Sheep red blood cells, WBC: White blood cell, ERK2: Extracellular signal-regulated kinase 2 immunomodulatory and anti-microbial effects. O. basilicum and its constituents shows anti-inflammatory and immunomodulatory property via improvement of inflammatory cells and inflammatory markers including IL-4, IL-10, TNF-α, IFN-γ and other cytokines. Anti-oxidant effects of this plant and its constituents were also shown by scavenging free radicals, reduction of oxidant agents and increase of anti-oxidant parameters. O. basilicum and its constituents modulated immune system via improvement of T-lymphocytes, and NK cells as well as inflammatory and anti-inflammatory cytokines, and Th1/Th2 balance. The wide range of study indicates that it is very beneficial for the improvement of current drugs and more work can be done to explore its advantage therapeutic potential in inflammatory, oxidative stress and immune dis-regulatory disorders.

Conclusion
The review articles showed anti-inflammatory, immunomodulatory and anti-oxidant effects of O. basilicum and its main constituents in various conditions which indicate possible therapeutic effects of the plant and its derivatives on inflammatory disorders, oxidative stress alignment and immune-dysregulatory diseases. However, more studies including clinical trials on the antiinflammatory, immunomodulatory and anti-oxidant effects of O. basilicum and its components are needed to performed before the plant and its constituents could be used for clinical purposes.